1. Field of the Invention
The present invention is in the fields of cell biology and biochemistry. The invention relates generally to cell and tissue culture medium compositions comprising metal binding compounds and/or transition element complexes comprising the metal binding compounds, and the use of such compounds and complexes. More specifically, the invention relates to compositions for cell and tissue culture comprising one or more transition element cations in a complex with a metal binding compound.
2. Related Art
Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial and in vitro environment. Characteristics and formulations of cell culture media vary depending upon the particular cellular requirements. Important parameters include osmolarity, pH, and nutrient compositions.
Medium formulations have been used to cultivate a number of cell types including animal, plant and bacterial cells. Cultivated cells have many uses including the study of physiological processes and the production of useful biological substances. Examples of such useful products include monoclonal antibodies, hormones, growth factors, enzymes and the like. Such products have many commercial and therapeutic applications and, with the advent of recombinant DNA technology, cells can be engineered to produce large quantities of these products. Cultured cells are also routinely used for the isolation, identification and growth of viruses which may be used as vectors and/or vaccines. Thus, the ability to cultivate cells in vitro is not only important for the study of cell physiology, but is also necessary for the production of useful substances which may not otherwise be obtained by cost-effective means.
Cell culture medium formulations have been well documented in the literature and a number of media are commercially available. In early cell culture work, medium formulations were based upon the chemical composition and physicochemical properties (e.g., osmolality, pH, etc.) of blood and were referred to as xe2x80x9cphysiological solutionsxe2x80x9d (Ringer, S., J. Physiol., 3:380-393 (1880); Waymouth, C., In: Cells and Tissues in Culture, Vol. 1, Academic Press, London, pp. 99-142 (1965);Waymouth, C., In Vitro 6:109-127 (1970)). However, cells in different tissues of the mammalian body are exposed to different microenvironments with respect to oxygen/carbon dioxide partial pressure and concentrations of nutrients, vitamins, and trace elements; accordingly, successful in vitro culture of different cell types may require the use of different medium formulations. Typical components of cell culture media include amino acids, organic and inorganic salts, vitamins, trace metals, sugars, lipids and nucleic acids, the types and amounts of which may vary depending upon the particular requirements of a given cell or tissue type.
Typically, cell culture medium formulations are supplemented with a range of additives, including undefined components such as fetal bovine serum (FBS) (10-20% v/v) or extracts from animal embryos, organs or glands (0.5-10% v/v). While FBS is the most commonly applied supplement in animal cell culture media, other serum sources are also routinely used, including newborn calf, horse and human. These types of chemically undefined supplements serve several useful functions in cell culture media (Lambert, K. J. et al., In: Animal Cell Biotechnology, Vol. 1, Spier, R. E. et al., Eds., Academic Press New York, pp. 85-122 (1985)). For example, these supplements provide carriers or chelators for labile or water-insoluble nutrients; bind and neutralize toxic moieties; provide hormones and growth factors, protease inhibitors and essential, often unidentified or undefined low molecular weight nutrients; protect cells from physical stress and damage; and provide carriers (e.g., transferrin and ceruloplasmin) for certain essential metal ions (e.g., Fe++ and Fe+++). Thus, serum and/or animal extracts are commonly used as relatively low-cost supplements to provide an optimal culture medium for the cultivation of animal cells.
Unfortunately, the use of serum or animal extracts in tissue culture applications has several drawbacks (Lambert, K. J. et al., In: Animal Cell Biotechnology, Vol 1, Spier, R. E. et al., Eds., Academic Prcs New York, pp. 85-122 (1985)). For example, the chemical composition of these supplements may vary between lots, even from a single manufacturer. In addition, supplements of animal or human origin may also be contaminated with adventitious agents (e.g., mycoplasma, viruses, and prions). These agents can seriously undermine the health of the cultured cells when these contaminated supplements are used in cell culture media formulations. Further, these agents may pose a health risk when substances produced in cultures contaminated with adventitious agents are used in cell therapy and other clinical applications. A major fear is the presence of prions which cause spongiform encephalopathies in animals and Creutzfeld-Jakob disease in humans.
The presence of serum in culture media can present additional difficulties. Cell surface chemistry, which is a critical portion of the in vitro microenvironment for many cell types, can be adversely modified via adsorption or incorporation of serum or extract proteins. The use of undefined components such as serum or animal extracts also prevents the true definition and elucidation of the nutritional and hormonal requirements of the cultured cells, thus eliminating the ability to study, in a controlled way, the effect of specific growth factors or nutrients on cell growth and differentiation in culture. Moreover, undefined supplements prevent the researcher from studying aberrant growth and differentiation and specific disease-related changes in cultured cells. Using cell culture media in the industrial production of biological substances, serum and animal extract supplementation of culture media can also complicate and increase the costs of the purification of the desired substances from the culture media due to the necessity of removing serum or extract proteins.
Serum-Free Media
To overcome the drawbacks of the use of serum or animal extracts, a number of serum-free media have been developed. These media, which often are specifically formulated to support the culture of a single cell type, incorporate defined quantities of purified growth factors, lipoproteins and other proteins usually provided by the serum or extract supplement. Since the components (and concentrations thereof) in such culture media are precisely known, these media arc generally referred to as xe2x80x9cdefined culture mediaxe2x80x9d and often as xe2x80x9cserum-free mediaxe2x80x9d or xe2x80x9cSFM.xe2x80x9d A number of SFM formulations are commercially available from, for example, Life Technologies, Inc. (Rockville, Md.) such as those designed to support the culture of endothelial cells, keratinocytes, monocytes/macrophages, fibroblasts, neurons, lymphocytes, chondrocytes, hematopoietic stem cells, embryonic stem cells, insect cells, CHO cells, Vero cells, 293 HEK cells, HeLa cells, PER-C6 (Human embryonal retinal epithelial cells), or hepatocytes.
SFM generally provide several distinct advantages to the user. For example, the use of SFM facilitates the investigation of the effects of a specific growth factor or other medium component on cellular physiology, which may be masked when the cells are cultivated in serum- or extract-containing media. In addition, SFM may contain much lower quantities of protein (indeed, SFM are often termed xe2x80x9clow protein mediaxe2x80x9d) than those containing serum or extracts, greatly simplifying and lowering the cost of purifying biological substances produced by cells cultured in SFM.
Some extremely simple SFM, which consist essentially of vitamins, amino acids, organic and inorganic salts and buffers have been used for cell culture. Such media (often called xe2x80x9cbasal mediaxe2x80x9d), however, are usually seriously deficient in the nutrition, hormone, or biological response modifier content required by most animal cells. Accordingly, most SFM incorporate into the basal media additional components to make the media more nutritionally or hormonally complex while attempting to maintain the serum-free and low protein content nature of the media. Examples of such components include serum albumin from bovine (BSA) or human (HSA), animal-derived lipids such as human Excyte (Bayer); sterols, etc., insulin, transferrin, and certain growth factors or hormones derived from natural (animal) or recombinant sources.
The use of such animal-derived supplements in cell culture media, however, also has certain drawbacks. For example, there is a risk that the culture medium and/or products purified from it may be immunogenic, particularly if the supplements are derived from an animal different from the source of the cells to be cultured. Thus, if biological substances to be used as therapeutics are purified from such culture media, certain amounts of these immunogenic proteins or peptides may be co-purified and may induce an immunological reaction, up to and including anaphylaxis, in an animal receiving such therapeutics.
To overcome this potential problem, supplements derived from the same species as the cells to be cultured may be used. For example, culture of human cells may be facilitated using HSA as a supplement, while media for the culture of bovine cells would instead use BSA. This approach, however, runs the risks of introducing contaminants and adventitious pathogens into the culture medium (such as HIV, Creutzfeld Jakob agent, or hepatitis viruses from HSA preparations, or Bovine Spongiform Encephalopathy prion from BSA preparations), which can obviously negatively impact the use of such media in the preparation of animal and human therapeutics. In fact, for such safety reasons, the biotechnology industry and government agencies are increasingly regulating, discouraging and even forbidding the use of cell culture media containing human or animal-derived products which may contain such pathogens.
Nothwithstanding the potential difficulties posed by the addition of animal derived supplements to cell culture media, such supplements are in routine use. One such supplement that is frequently added to defined media is transferrin. Transferrin functions in vivo to deliver iron to cells. The mechanism of iron uptake by mammalian cells has been reviewed (Qian, Z. M. and Tang, P. L. (1995) Biochim.Biophys.Acta 1269, 205-214). As iron is required as a co-factor in numerous metabolic processes including energy generation and oxidative respiration, serum-free media are often supplemented with transferrin in order to deliver the requisite iron for the successful cultivation of most cells in vitro. Concern about various potential adventitious agents in preparations of transferrin has stimulated a search for other natural iron carrier compounds which can be used as a substitute for transferrin. This search is complicated by the fact that the natural iron carriers are often derived from serum and thus are subject to the above-described limitations of serum supplementation.
Metal Binding Compounds
To overcome the limitations of using naturally derived metal carriers, certain metal binding compounds are being explored for use in supplying metals, particularly zinc, iron, manganese and magnesium, to cultured cells. Simple carriers such as chelating agents (e.g., EDTA) and certain acids or salts thereof (e.g., citrate, picolinate, and derivatives of benzoic acid or hydroxamic acid) have been shown to be useful in certain serum-free growth media (see U.S. Pat. Nos. 5,045,454 and 5,118,513; Testa et al., Brit. J. Haematol. 60:491-502, (1985); Ganeshaguru et al., Biochem. Pharmacol. 29:1275-1279 (1980); White et al., Blood 48:923-929 (1976)).
Although these references disclose some metal carriers, the interpretation of the data is complicated by several experimental factors. The data were gathered from a limited number of cell lines and show results of a single passage. In addition, the media were supplemented with serum. Serum inherently contains transferrin and other potential iron carriers. There is a xe2x80x9ccarry-over effectxe2x80x9d on growth of cells which have been cultured in serum-supplemented medium, even after one or two passages in the absence of serum or transferrin (see, for example, Keenan, J. and Clynes, M. (1996) In Vitro Cell Dev.Biol-Animal 32, 451-453). Other known metal binding compounds have been used medicinally to remove iron from the body and not for delivery. Unfortunately, many of these simple iron chelating compounds do not provide sufficient iron availability to, or uptake by, cultured cells.
Thus, there remains a need in the art for compounds capable of delivering transition metals to cells cultured in vitro. More specifically, there exists a need in the art for iron carriers for use in serum-free, low-protein culture medium which are suitable for cultivation of eukaryotic and prokaryotic cells. Such a medium formulation will facilitate studies of the effects of growth factors and other stimuli on cellular physiology, will allow easier and more cost-effective purification of biological substances produced by cultured animal cells in the biotechnology industry, and most importantly will eliminate the risk of the introduction of adventitious animal and human pathogens. The current invention provides such a variety of compounds or specifically iron carriers for an animal cell culture medium formulation.
The present invention meets the need in the art for compounds capable of delivering transition metals to cells cultured in vitro by providing culture media that comprise one or more non-animal-derived metal-binding compounds which may be used to provide essential metals (e.g., Fe++ or Fe+++ ions) to cells. The compounds provided by the invention comprise a metal binding compound which may be added to a medium alone or may be complexed with one or more transition element ions prior to addition to the medium. The compounds described herein were used to facilitate the delivery of transition metals to cells cultured in vitro. In particular, the compounds of the present invention were selected for their ability to deliver iron and to replace transferrin. The compounds were tested in the absence of transferrin for at least three sub-cultures. These compounds have been tested for their ability to support the growth of at least three commonly used cell lines: CHO, Sp2/0, and 293 HEK cells.
The present invention is also directed to compositions, particularly cell culture media, comprising one or more metal binding compounds of the invention and/or one or more transition elements in a complex with one or more metal binding compounds of the invention. Such cell culture media of the invention may be used to grow or cultivate plant cells, animal cells (particularly human cells), insect cells, bacterial cells, yeast cells and more generally any type of eukaryotic or prokaryotic cells. Thus, the metal binding compounds of the present invention may be added to any type or kind of culture media, and are preferably used to replace naturally derived metal carriers (e.g., animal derived proteins or extracts such as transferrin) in such media. The invention is also directed to methods of use of such compositions, including, for example, methods for the cultivation of eukaryotic cells, particularly animal cells, in vitro. The invention also relates to compositions comprising such culture media and one or more cells, and to kits comprising one or more of the above-described compositions.
Culture media of the invention are preferably serum-free, and may comprise at least one metal binding compound and/or at least one transition element complex, said complex comprising at least one transition element or a salt or ion thereof, in a complex with at least one metal-binding compound, wherein the medium is capable of supporting the cultivation of a cell in vitro in the absence of naturally derived metal carriers such as transferrin or other animal derived proteins or extracts. The metal binding compound may be in a complex with a transition metal prior to addition of the metal binding compound to the medium. In other embodiments, the metal binding compound is not in a complex with a transition metal prior to addition of the metal binding compound to the media.
According to one aspect of the invention, a transition element is preferably selected from the group consisting of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, rubidium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, and actinium, or salts or ions thereof, and is preferably an iron salt. Suitable iron salts include, but are not limited to, FeCl3, Fe(NO3)3 or FeSO4 or other compounds that contain Fe+++ or Fe++ ions.
Metal binding compounds of the present invention include any molecules which may interact with or bind with transition elements and facilitate the uptake by cells. Such interaction/binding may be covalent or non-covalent in nature. The metal-binding compound used in this aspect of the invention is preferably selected from the group consisting of a polyol, a hydroxypyridine derivative, 1,3,5-N,Nxe2x80x2,Nxe2x80x3-tris(2,3-dihydroxybenzoyl)aminomethylbenzene, ethylenediamine-N,Nxe2x80x2-tetramethylenephosphonic acid, trisuccin, an acidic saccharide (e.g. ferrous gluconate), a glycosaminoglycan, diethylenetriaminepentaacetic acid, nicotinic acid-N-oxide, 2-hydroxy-nicotinic acid, mono-, bis-, or tris-substituted 2,2xe2x80x2-bipyridine, a hydroxamate derivative (e.g. acetohydroxamic acid), an amino acid derivative, deferoxamine, ferrioxamine, iron basic porphine and derivatives thereof, DOTA-lysine, a texaphyrin, a sapphyrin, a polyaminocarboxylic acid, an xcex1-hydroxycarboxylic acid, a polyethylenecarbamate, ethyl maltol, 3-hydroxy-2-pyridine, and IRC011. In one preferred embodiment, the metal-binding compound is a polyol such as sorbitol or dextran, and particularly sorbitol. In a related embodiment, the metal-binding compound is a hvdroxvpvridine derivative, such as 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone, 3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid4-one, 1-hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid-4one, 1-methyl-3-hydroxypyrid-2-one, (3-hydroxy-2(1H)-pyridinone, ethyl maltol or pyridoxal isonicotinyl hydrazone, and is preferably 2-hydroxypyridine-N-oxide. In particularly preferred embodiments according to this aspect of the invention, the transition metal complex may be a sorbitol-iron complex or 2-hydroxypyridine-N-oxide-iron complex. The metal binding compounds of the present invention may also bind divalent cations such as Ca++ and Mg++.
In a related aspect, the invention relates to cell culture media comprising at least one metal binding compound capable of binding or interacting with one or more transition elements and further comprising one or more ingredients selected from the group of ingredients consisting of at least one amino acid (such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine or L-valine, N-acetyl-cysteine), at least one vitamin (such as biotin, choline chloride, D-Ca++-pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine or vitamin B12), at least one inorganic salt (such as a calcium salt, CuSO4, FeSO4,Fe(NO3)3, FeCl3, KCl, a magnesium salt, a manganese salt, sodium acetate, NaCl, NaHCO3, Na2HPO4, Na2SO4, a selenium salt, a silicon salt, a molybdenum salt, a vanadium salt, a nickel salt, a tin salt or a zinc salt), adenine, ethanolamine, D-glucose, at least one cytokine, heparin, hydrocortisone, lipoic acid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, tri-iodothyronine, dextran sulfate, Pluronic F68, and thymidine. The culture media of the present invention may optionally include a buffeting agent. Suitable buffering agents include, but are not limited to, N-[2-hydroxyethyl]-piperazine-Nxe2x80x2-[2-ethanesulfonic acid] (HEPES), MOPS, MES, phosphate, carbonate and other buffering agents suitable for use in cell culture applications. A suitable buffering agent is one that provides buffering capacity without substantial cytotoxicity to the cells cultured. The selection of suitable buffering agents is within the ambit of ordinary skill in the art of cell culture.
In another aspect, the invention relates to a metal binding compound capable of binding a transition element in a cell culture medium comprising one or more of the ingredients selected from the group: adenine, ethanolamine, D-glucose, N-[2-hydroxyethyl]piperazine-Nxe2x80x2-[2-ethanesulfonic acid] (HEPES), hydrocortisone, lipoic acid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, tri-iodothyronine, thymidine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, N-acetyl-cysteine, biotin, choline chloride, D-Ca++-pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine, vitamin B12, a calcium salt, recombinant insulin, dextran sulfate, Pluronic F68, CuSO4, FeSO4, FeCl3, KCl, a magnesium salt, a manganese salt, sodium acetate, NaCl, NaHCO3, Na2HPO4, Na2SO4, a selenium salt, a silicon salt, a molybdenum salt, a vanadium salt, a nickel salt, a tin salt, and a zinc salt, and one or more transition element complexes comprising at least one transition element or a salt or ion thereof complexed to at least one metal-binding compound, wherein each ingredient is present in an amount which supports the cultivation of a cell in vitro. Preferred transition elements, metal-binding compounds, and transition element complexes for use in this aspect of the invention include those described in detail herein.
In another aspect, the invention relates to a cell culture medium obtained by combining a medium with at least one metal binding compound and/or at least one transition element complex, said complex comprising at least one transition element or a salt or ion thereof complexed to at least one metal-binding compound, wherein said medium is capable of supporting the cultivation of a cell in vitro. Preferred transition elements, metal-binding compounds, and transition element complexes for use in this aspect of the invention include those described in detail herein.
According to the invention, a medium suitable for use in forming the cell culture media of the invention may comprise one or more ingredients, and may be obtained, for example, by combining one or more ingredients selected from the group consisting of adenine, ethanolamine, D-glucose, heparin, a buffering agent, hydrocortisone, lipoic acid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, tri-iodothyronine, thymidine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, Lysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, N-acetyl-cysteine, biotin, choline chloride, D-Ca++-pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine, vitamin B12, dextran sulfate, Pluronic F68, recombinant insulin, a calcium salt, CuSO4, FeSO4, FeCl3, Fe(NO3)3, KCl, a magnesium salt, a manganese salt, sodium acetate, NaCl, NaHCO3, Na2HPO4, Na2SO4, a selenium salt, a silicon salt, a molybdenum salt, a vanadium salt, a nickel salt, a tin salt and a zinc salt, wherein each ingredient is added in an amount which supports the cultivation of a cell in vitro.
According to the invention, the culture media provided by or obtained by the invention may be 1xc3x97medium formulations, or may be concentrated medium formulations, for example 10xc3x97, 20xc3x97, 25xc3x97, 50xc3x97, 100xc3x97, 500xc3x97, or 1000xc3x97medium formulations.
Both eukaryotic and prokaryotic cells may be cultivated in the media provided or obtained by the invention, including but not limited to mammalian cells (including human cells), bird cells, insect cells, fish cells, amphibian cells, and reptile cells, any or all of which may be normal cells or abnormal cells (such as transformed cells, established cells, or a cells derived from a diseased tissue sample). Cells may also include plant cells or microbial cells such as bacteria and lower eukaryotic cells such as fungi and yeast.
In another aspect, the invention relates to methods of cultivating cells, such as those cells described above, comprising (a) contacting the cell with the cell culture media of the invention; and (b) cultivating the cell under conditions suitable to support cultivation or growth of the cell.
In another aspect, the invention relates to kits for the cultivation of a cell in vitro. Kits according to one such aspect of the invention may comprise one or more of the culture media of the invention, one or more metal binding compounds, and/or one or more transition element complexes. Kits according to another aspect of the invention may comprise one or more cell culture media (one of which may be a basal medium) and at least one metal binding compound and/or at least one transition element complex, said complex comprising at least one transition element or a salt or ion thereof complexed to at least one metal-binding compound. Preferred transition elements, metal-binding compounds, and transition element complexes for use in the kits according to this aspect of the invention include those described in detail herein.
In another aspect, the invention relates to compositions comprising the culture media of the invention and one or more cells, including those cells described above.
Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims.